The present invention relates to compounds that inhibit glycosidases and HIV proteases. More particularly, the present invention relates to seven-membered hydroxyiminocyclitols, also known as hydroxyazepanes, which have inhibitory activity toward glycosidases and hiv protease and to chemical and enzymatic/chemical methods for synthesizing such compounds.
Glycosidases are involved in the processing and synthesis of complex carbohydrates which are essential for various biological recognition processes. Because of their multifaceted biological importance, these enzymes are often targeted for inhibition. (Look et al. Acc. Chem. Res. 1993, 26, 182; Winchester et al. Glycobiology 1992, 2, 1991; Wong et al. Angew. Chem. Int. Ed. Engl. 1995, 34, 521; Legler et al. Adv. Carbohydr. Chem. Biochem. 1990, 48, 319; Sinnott et al. Chem. Rev. 1990, 90, 1171; Kirby et al. Adv. Phys. Org. Chem. 1994, 29, 87; Jacob et al. Curr. Opin. Struct. Biol. 1995, 5, 605.
Glycosidases and aspartyl proteases share a common mechanism in catalysis, i.e. both utilize two carboxyl groups as general acid and general base in the hydrolytic reactions (Wlodawer et al. Science 1989, 245, 616; Hyland et al. Biochemistry 1991, 30, 8454; Reviews on the mechanisms of glycosidases: Sinnott et al. Chem. Rev. 1990, 90, 1171; Legler et al. Adv. Carb. Chem. Biochem. 1990, 48, 319; Withers et al. Pure and Appl. Chem. 1995, 67, 1673).
Five- and six-membered azasugar families have been characterized as mimics of the transition state of the enzymatic reactions of glycosidases and aspartyl proteases. (Withers et al. J. Am. Chem. Soc. 1988, 110, 8551; Ganem et al. J. Am. Chem. Soc. 1991, 113, 8984; Papandreou et al. J. Am. Chem. Soc. 1993, 115, 11682; Fotsch et al. Tetrahedron Lett. 1994, 35, 3481; Jeong et al. J. Am. Chem. Soc., 1996, 118, 4227.) Five- and six-membered iminocyclitols, for example, have been used as transition-state analog inhibitors of glycosidases (Elbein et al. Annu. Rev. Biochem. 1987, 56, 497; Wichester et al. Glycobiology 1992, 2, 199; Look et al. Acc. Chem. Res. 1993, 26, 182; Jacob et al. Current Opinion in Structural Biology 1995, 5, 605) and various peptide isosteres, including those with C2-symmetry, have been developed as inhibitors of the HIV (Human Immunodeficiency Virus) protease (Erickson et al. Perspectives in Drug Discovery and Design 1993, 1, 109.)
Unlike five and six-membered iminocyclitols, the biological activities of seven-membered iminocyclitols, also known as hydroxyazepanes, are largely uncharacterized. One hydroxyazepane was reported to have no inhibitory activity against xcex1-mannosidase. (Farr et al. Tetrahedron 1994, 50, 1033.) These heterocycles are conformationally more flexible than the corresponding six- and five-membered counterparts and may adopt the half-chair or pseudo-chair structure to mimic the transition state of the enzymatic glycosidic cleavage. (Qian et al. Bioorg. Med. Chem. Lett. 1996, 6, 1117.)
Conventional synthetic methods for producing hydroxyazepanes require lengthy linear chemical routes with multiple steps in overall low yields. (Paulsen et al. Chem. Ber 1967, 100, 512; Poitout et al. Tetrahedron Lett. 1994, 35, 3293; Lohray et al. J. Org. Chem. 1995, 60, 5958; Farr et al. Tetrahedron 1994, 50, 1033.)
What is needed is hydroxyazepanes having inhibitory activity with respect to glycosidases and aspartyl proteases and chemical or chemo/enzymatic methods for synthesizing hydroxyazepanes in high yields with a small number of synthetic steps.
One aspect of the invention is directed to methodologies for the chemical or chemo/enzymatic synthesis of seven-membered iminocyclitols which are also known as hydroxyazepanes. A series of hydroxyazepanes is obtained either by chemoenzymatic or chemical synthesis. The compounds display significant activity as glycosidase inhibitors, with Ki values from moderate to low micromolar range. The 3-benzyl and 3,6-dibenzyl derivatives of these hydroxyazepanes, viz. compounds 60a, 60b, 64a, 64b, 65a, and 65b (FIGS. 8 and 20), inhibit the mechanistically related HIV protease.
In a first mode, a chemo/enzymatic methodology is employed for synthesizing hydroxyazepanes. In the first step, there is an addition of (xc2x1)-3-azido-2-hydroxypropanaldehyde with dihydroxyacetone phosphate (DHAP) in the presence of a DHAP dependent aldolase to produce a 6-azido-3,4,5-trihydroxy-2-hexanone-1-phosphate intermediate. In the second step, there is an hydrolysis of the 6-azido-3,4,5-trihydroxy-2-hexanone-1-phosphate intermediate to produce a polyhydroxy 6-deoxy-6-azido ketose by treatment with acid phosphatase. In the third step, there is an isomerization of the ketose to a 6-azido-6-deoxyaldopyranose by treatment with an isomerase. The fourth and final step comprises cyclization of the pyranose to a seven membered hydroxyazepane using reductive amination conditions on the C6 azide moiety of the pyranose with hydrogen and a catalyst.
Alternative modes include several novel chemical syntheses of hydroxyazepanes which involve chemical manipulations of aldopyranoses protected as benzyl glycosides or diisopropylidene ethers. All of the chemical syntheses involve the use of a Pd-mediated reductive amination conditions for ring expansion of the six membered pyranose to form the seven membered azepane. Examples of this chemical approach show that D-galactose can be used to obtain a meso-3,4,5,6-tetrahydroxyperhydroazepine; D-mannose can be used to obtain a derivative with a C2 symmetry axis and N-acetylglucosamine can be used to obtain a 6-acetamidoiminocyclitol.
More particularly, one mode of the invention is directed to a method for producing a tetrahydroxyazepane. In this mode, there is an initial addition reaction of 3-azido-2-hydroxypropanaldehyde with dihydroxyacetone phosphate using an aldolase for producing a 6-azido-3,4,5-trihydroxy-2-hexanone-1-phosphate intermediate with the formula: 
Then the above 6-azido-3,4,5-trihydroxy-2-hexanone-1-phosphate intermediate is hydrolyzed with acid phosphatase for producing a polyhydroxy 6-deoxy-6-azido ketose intermediate with the formula: 
Then the above polyhydroxy 6-deoxy-6-azido ketose intermediate is isomerized with an isomerase for producing a 6-azido-6-deoxyaldose intermediate with the formula: 
Then the above 6-azido-6-deoxyaldose intermediate is cyclized using reductive amination conditions with hydrogen and a catalyst for producing the tetrahydroxyazepane with the formula: 
An alternative mode of the invention is directed to another method for synthesizing a tetrahydroxyazepane. The 6-hydroxyl position of a 6-hydroxy-1,2,3,4-protected monosaccharide is activated with an activating agent for producing an activated 6-hydroxy-1,2,3,4-protected monosaccharide with the formula: 
wherein R1 is selected from the group consisting of benzyl and isopropylidene; R2 is selected from the group consisting of tosylate and (P(Phenyl)3)+. Then the above activated 6-hydroxy-1,2,3,4-protected monosaccharide is admixed with an azide donor for producing a 6-azido-6-deoxy-1,2,3,4-protected monosaccharide with the formula: 
wherein R1 is selected from the group consisting of benzyl and isopropylidene. Then, the above protected 6-azido-6-deoxy-1,2,3,4-protected monosaccharide is deprotected with a deprotecting agent for producing a 6-azido-6-deoxysugar with the formula: 
Then, the above 6-membered ring of the 6-azido-6-deoxysugar is expanded under reductive amination conditions with hydrogen and a catalyst for producing the tetrahydroxyazepane with the formula: 
In a preferred mode, the 6-azido-6-deoxysugar is then converted into a 3-methoxy-tri-hydroxyazepane with additional steps. The 6-azido-6-deoxysugar is glycosylated with a glycosylating agent for producing a 6-azido-6-deoxyglycoside with the formula: 
wherein R1 is selected from the group consisting of methyl, propyl and benzyl. Then, the 3 and 4 hydroxyl positions on the above 6-azido-6-deoxyglycoside is blocked with a protecting agent for producing a 1,3,4,6-blocked 2-hydroxy-6-azido-6-deoxyglycoside with the formula: 
wherein R1 is selected from the group consisting of isopropylidene and benzylidene. Then, the 2 hydroxyl position on the above 1,3,4,6-blocked 2-hydroxy-6-azido-6-deoxyglycoside is methylated with a methylating agent for producing a 1,3,4,6-blocked 2-methoxy-6-azido-6-deoxyglycoside with the formula: 
Then, the above 1,3,4,6-blocked 2-methoxy-6-azido-6-deoxyglycoside is deprotected with a deprotecting agent for producing a 2-methoxy-6-azido-6-deoxyglycoside with the formula: 
Then, the 6-membered ring of the above 2-methoxy-6-azido-6-deoxyglycoside is expanded using reductive amination conditions with hydrogen and a catalyst for producing the tetrahydroxyazepane with the formula: 
In an alternative mode of the invention, a 2-acetamido-3,4,5-trihydroxyazepane is synthesized by another method. The 6-hydroxyl position on a 2-acetamido-pyranose monosaccharide is activated with an activating agent for producing an activated 2-acetamido-pyranose monosaccharide with the formula: 
wherein R1 is selected from the group consisting of tosylate and (P(Phenyl)3)+. Then, the above activated 2-acetamido-pyranose monosaccharide is admixed with an azide donor for producing a 6-azido-2-acetamido-pyranose monosaccharide with the formula: 
Then, the 6-membered ring of the above 6-azido-2-acetamido-pyranose monosaccharide is expanded using reductive amination conditions with hydrogen and a catalyst for producing the 2-acetamido-3,4,5-trihydroxyazepane with the formula: 
In an alternative mode of the invention, a 2-hydroxymethyl-3,4-dihydroxy-5-methoxypiperidine is synthesized by another method. In the first step, there is an addition of 3-azido-2-hydroxypropanaldehyde with dihydroxyacetone phosphate using an aldolase for producing a 6-azido-5-methoxy-3,4-dihydroxy-2-hexanone-1-phosphate intermediate with the formula: 
Then, the above 6-azido-5-methoxy-3,4-dihydroxy-2-hexanone-1-phosphate intermediate is dephosphorylated with acid phosphatase for producing a 6-azido-5-methoxy-1,3,4-trihydroxy-2-hexanone intermediate with the formula: 
Then, the above 6-azido-5-methoxy-1,3,4-trihydroxy-2-hexanone intermediate is cyclized using reductive amination conditions with hydrogen and a catalyst for producing the 2-hydroxymethyl-3,4-dihydroxy-5-methoxypiperidine with the formula: 
In each of the above alternative modes, the aldolase is selected from the group consisting of rhamnulose-1-phosphate aldolase, rabbit muscle aldolase, fructose-1,6-diphosphate aldolase and fucose aldolase. The isomerase is selected from the group consisting of rhamnose isomerase, fucose isomerase, glucose isomerase and galacatose isomerase. The catalyst is selected from the group consisting of palladium on carbon and platinumon carbon. The activating agent is selected from the group consisting of tosyl chloride and diethylazodicarboxylate with triphenylphosphine. The azide donor is selected from the group consisting of sodium azide and diphenylphosphorylazide. And, the deprotecting agent is selected from the group consisting of hydrogen with palladium on carbon, HCl/water combination and acetic acid/water combination.
In another aspect of the invention, tetrahydroxyazepane is synthesized according to the following method. A 6-azido-6-deoxyaldose intermediate with the formula: 
is cyclized using reductive amination conditions with hydrogen and a catalyst for producing the tetrahydroxyazepane with the formula: 
Another aspect of the invention is directed to active compounds represented by the following structures: 